Agriculture produces the vast majority of the world’s food supply, and in last century
the global food production has grown at a huge rate mainly from the increased yields
resulting from greater inputs of insecticides and other technologies. Meanwhile overuse or
improper use of insecticides and other agrochemicals has raised issues about related
environment and health costs, with current legislation promoting sustainable agriculture, in
which scenario, the development of environmentally sustainable strategies is mandatory for
research programs regarding pest control. The entomopathogenic mitosporic ascomycetes
(EMAs) and in particular the genus Metarhizium have shown great success in the control of
insect pests due to their contact mode of action, natural presence in the ecosystems and
their ability to secrete compounds with insecticidal activity, and even, they comply with the
security requirements for human health and environment, whereas information about the
fate of their secondary metabolites in the food chain and their risk to human and animal
health is still scarce. There is a need to develop and validate analytical methods with high
sensitivity for metabolite determination at low concentrations in different biological
matrices. Destruxin A is one of the major secondary metabolite produced by the genus
Metarhizium spp., but the lack of studies concerning destruxin A production is most likely
the biggest obstacle for registration of new fungal strains. The main goal of this research has
been to develop new tools for destruxin detection and quantification and to investigate the
fate of destruxin A in the trophic chain.
In chapter II, destruxin production for Metarhizium strains BIPESCO5, EAMa 01/58-
Su, ARSEF 23 and ART 2825 was determined with an improved method of ultra-high
performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which
has shown high precision in the detection and quantification of destruxins in four culture
media (CM, MM, CN2, OSM) representing different stress conditions. Every 3 days samples
were taken for analysis over 18 days that allowed detecting 15 destruxins, with destruxin A
and B as the most abundant. However, significant differences among strains in destruxin
production were detected, and for each strain, destruxin production was highly dependent
on culture medium. In chapter III, endophytic colonisation and destruxin A production on potato plants
were monitored at 24, 48, 72, 96 and 120 h after inoculation with Metarhizium brunneum
strains (BIPESCO5 and EAMa 01/58-Su), which showed that the concentration of destruxin A
in plant tissues was very low compared to the colonisation levels. Although a similar
colonisation was observed for both strains, there were differences in percentages in
different parts of the plants, with the higher values occurring in the leaves at 96 h for EAMa
01/58-Su (83.3 %) and BIPESCO5 (81.6 %), and the lower ones, 10.0-13.3 %, observed in
tuber and root at 72, 96 and 120 h post-inoculation for both strains. For strain EAMa 01/58-
Su, destruxin A was quantified at 24 h (2.49 ± 1.7 and 2.0 ± 1.4 μg/kg, respectively), and the
same concentration was found in both tuber and root at 96 h (2.5 ± 1.7 μg/kg); for
BIPESCO5, the concentrations differed in tuber at 24 h and in root at 48 h (6.8 ± 4.8 and 2.1
± 1.4 μg/kg, respectively).
In chapter IV, the dynamic of fungal growth and secretion of destruxin A by strains
BIPESCO5 and EAMa 01/58-Su of Metarhizium brunneum Petch. during the infection process
of larvae of the model insect Galleria mellonella L. (Lepidoptera; Pyralidae) was monitored
for the first time. Data showed that destruxin A secretion was parallel to the fungal growth
of EAMa 01/58-Su but not coupled with that for BIPESCO5. EAMa 01/58-Su and BIPESCO5
strains secreted destruxin A from days 2 to 6 and from day 2 to day 5 post treatment,
respectively. For EAMa 01/58-Su and BIPESCO5, the maximum titer in the host on day 4
after treatment was 0.369 and 0.06 μg/larva, respectively, and throughout the pathogenic
process, the production was 0.6 and 0.09 μg/larva, respectively.
In chapter V, predator-prey bioassays were performed to evaluate the behavior and
survival of larvae of the generalist predator Chrysoperla carnea (Stephens) (Neuroptera;
Chrysopidae) when feeding on larvae of the polyphagous pest Spodoptera littoralis (Boisd.)
(Lepidoptera; Noctuidae) challenged by M. brunneum BIPESCO5 and EAMa 01/58-Su strains.
In addition, ecotoxicological studies based on HPLC-MS were performed to monitor the fate
of destruxin A in the prey-predator system. The maximum concentration of destruxin A
produced by the BIPESCO5 strain was on day 4 after treatment with a value of 0.000054
μg/insect (approx 0.014 μg/g), and for EAMa 01/58-Su was on day 5 with a value of 0.00012
μg/insect (approx 0.031 μg/g), whereas the metabolite was no detected in C. carnea larvae.
The percentage of lacewings feeding on S. littoralis larvae 24 hour-post infection was 96.6, 75.0, and 65.0 % for the control, EAMa 01/58-Su, and BIPESCO5 treatments, respectively,
whereas 5 days-post infection armyworm larvae were consumed by only 38.3 % of the
control lacewings and 33.3 % of the EAMa 01/58-Su and BIPESCO5 treatment groups. C.
carnea larvae feeding on 24 h-post infection armyworm larvae preyed 5.6, 2.2 and 2.3
larvae for the control, EAMa 01/58-Su and BIPESCO5 treatments, respectively, whereas
those predator larvae feeding on 5 days-post infection armyworm larvae preyed on only one
per capita larva. It showed that the M. brunneum treatments against S. littoralis larvae were
safe for C. carnea due to both the lack of fungus-related mortality in the predator and the
lack of movement of destruxin A from the prey to the predator.
Notably in chapters IV and V, in both M. brunneum strains, mortality from other
causes was higher than mortality with fungal outgrowth. However, destruxin A secretion
was higher for EAMa 01/58-Su than for BIPESCO5. These results suggested that destruxin A
could be a virulence factor for EAMa 01/58-Su strain, whereas for BIPESCO5, the virulence
could require the involvement of other factors as well as destruxin A during the infection
process. The results obtained provide valuable analytical methods for carrying out risk
assessments on the use of EMAs. In addition, results indicate that their use poses a little
potential hazard to human and animal health and the environment.